Project Summary The goal of this application is to elucidate properties of human leukemia stem cells (LSCs) that will lead to improved strategies for therapeutic targeting. Specifically, in the context of acute myelogenous leukemia (AML), LSCs are thought to reside at the heart of disease, driving progression and relapse. While many drugs have activity in AML, few if any clinically approved agents are known to efficiency eradicate LSCs. Thus, our studies have focused on the fundamental property of energy metabolism as a potential entry point for developing novel therapies. It has become increasingly clear that normal hematopoietic stem cells (HSCs) display unique metabolic properties that distinguish them from other cells in the hematopoietic system. We propose that AML stem cells will be similarly unique and employ metabolic mechanisms that are distinct from bulk AML cells as well as normal stem/progenitor cells. Indeed, our preliminary data show that like normal HSCs, most LSCs reside in a state of relatively low reactive oxygen species (termed ?ROS-low?). Further, we show that the metabolic sensor AMPK is preferentially activated in the ROS-low LSC population, and is responsible for modulating energy metabolism as reflected in the use of fatty acid oxidation as well as steady- state levels of NADPH. Importantly, genetic inhibition of AMPK is sufficient to dramatically reduce the functional ability of LSCs. These data lead us to hypothesize that LSCs preferentially reside in a distinct metabolic condition characterized by a ROS-low status and activation of AMPK. Further, we propose that modulation of AMPK (and related pathways) can be employed to ?push? LSCs out of the ROS-low state, thereby reducing their fitness and/or rendering them more susceptible to targeting by available therapeutic agents. To test these theories, we will perform a detailed metabolomic analysis of primary human AML specimens in order to examine relative utilization of energy pathways in primitive cell types. These studies will focus in particular on the role of AMPK. We will also perform drug discovery/development studies to create improved small molecule inhibitors of AMPK. Taken together, the proposed studies will elucidate the key metabolic pathways that control energy metabolism in LSCs and identify novel strategies for therapeutic targeting of the LSC population.